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Unicellular Organisms: Eubacteria, Archea, Yeast Lecture 27, Chapter 21 May 13, 2004 Jeff Esko. Overview. General structure of bacterial cell walls Structure, function and assembly of peptidoglycan (murein) Periplasmic  -glucans (MDO) Lipopolysaccharide (LPS) - endotoxin

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Unicellular organisms eubacteria archea yeast lecture 27 chapter 21 may 13 2004 jeff esko

Unicellular Organisms: Eubacteria, Archea, Yeast Lecture 27, Chapter 21May 13, 2004Jeff Esko


Overview
Overview

  • General structure of bacterial cell walls

    • Structure, function and assembly of peptidoglycan (murein)

    • Periplasmic -glucans (MDO)

    • Lipopolysaccharide (LPS) - endotoxin

    • Capsular polysaccharides - mimicry

  • Archea glycoproteins - a new area

  • Yeast glycosylation and cell walls


Capsule

LPS

PG

MDO

Gram-negative bacteria cell wall


Peptidoglycan

A

A

A

A

D

D

D

D

G

G

G

G

A

A

A

A

A

A

A

A

G

G

G

G

D

D

D

D

A

A

A

A

A

A

A

D

D

D

G

G

G

A

A

A

A

A

A

A

G

G

G

G

D

D

D

D

A

A

A

A

A

A

A

A

D

D

D

D

G

G

G

G

A

A

A

A

A

A

A

A

G

G

G

G

D

D

D

D

A

A

A

A

Peptidoglycan

[GlcNAcb1,4MurNAcb1,4]n

Heijenoort (2001) Glycobiology 11:25R


Murein glycopeptide

C

H

O

H

C

H

O

H

2

2

O

O

O

O

O

O

O

H

C

C

H

N

H

A

c

N

H

A

c

3

O

C

L

-

A

l

a

D

-

G

l

u

D

A

P

D

-

A

l

a

D

-

A

l

a

Murein Glycopeptide

Notice D-amino acids

DAP = diaminopimelic acid


Peptidoglycan1

A

A

A

A

D

D

D

D

G

G

G

G

A

A

A

A

A

A

A

A

G

G

G

G

D

D

D

D

A

A

A

A

A

A

A

D

D

D

A

A

A

A

G

G

G

A

A

A

A

A

A

A

G

G

G

G

D

D

D

D

A

A

A

A

A

A

A

A

D

D

D

D

G

G

G

G

A

A

A

A

A

A

A

A

G

G

G

G

D

D

D

D

A

A

A

A

Peptidoglycan

The final step involves cleavage between the D-Ala-D-Ala unit and transpeptidation to the amino group of DAP of another unit




Peptidoglycan biosynthesis

P

Peptidoglycan Biosynthesis

Undecaprenyl

phosphate


MDO

Gram-negative bacteria cell wall


Membrane derived oligosaccharides mdo

b2

b2

b2

b2

b2

b2

b2

Membrane Derived Oligosaccharides (MDO)

  • Branched -glucans

  • Represent about 1-5% of dry weight

  • Charged substituents act as an osmolyte and protects the inner membrane against the large difference in osmolarity inside the cell compared to outside the cell

+ Phosphoethanolamine

+ Phosphoglycerol

+ Succinate


LPS

Gram-negative bacteria cell wall


Lipopolysaccharide lps
Lipopolysaccharide (LPS)

  • LPS consists of three domains

    • Lipid A, otherwise known as endotoxin

    • Core region composed of KDO (K), heptoses (H), and hexoses (open hexagons)

    • Highly variable outer O-antigen region


H

O

O

H

H

O

H

O

H

O

U

r

i

d

i

n

e

U

r

i

d

i

n

e

N

H

AcN

O

H

O

H

O

H

O

H

H

O

H

O

O

O

O

O

O

O

O

P

P

P

P

O

O

O

C

O

O

O

O

O

  • Initiates by acylation of UDP-GlcNAc at C3, followed by N-deacetylation, and N-acylation

Wyckoff et al. (1998) Trends Microbiol. 6:154


Lipid a assembly

O

H

O

H

O

H

C

H

2

O

O

O

O

H

O

O

O

H

O

H

O

O

O

O

O

O

H

O

O

U

r

i

d

i

n

e

N

H

P

P

N

H

N

H

O

P

O

C

O

O

O

C

O

O

C

O

O

N

H

P

O

O

H

O

H

O

O

O

C

O

O

H

O

O

O

H

H

O

H

O

H

O

H

O

H

O

H

O

H

O

H

O

Lipid A Assembly

+

Diacylglucosamine-1-P condenses with another molecule of UDP-diacylglucosamine to form the tetraacyl disaccharide core


Lipid a assembly1
Lipid A Assembly

  • KDO transferases initiate the formation of the core

  • Additional C12 fatty acids added to -hydroxy groups (wax)

  • Lipid A translocates to the outer leaflet of the outer membrane by msbA (ABC transporter)

Doerrler et al. (2001) J Biol Chem. 276:11461


Lipid a biology
Lipid A Biology

  • Lipid A, the heat stable endotoxin of gram negatives

  • Resistant strains of mice defined a locus, lps, which was positionally cloned.

  • lps turned out to be homologous to toll receptors in Drosophila, which were known to be involved in innate immunity to fungal infection

  • lps turns out to beTlr4, a member of a family of signaling receptors (10 members known).

    • Tlr4 binds to Lipid A.

    • Tlr2 apparently binds and responds to muramyl-dipeptide




Core region contains unusual sugars

C

H

O

H

2

C

H

O

H

2

H

O

H

C

C

H

O

H

O

C

O

O

H

O

H

O

O

H

O

H

O

H

O

H

O

H

O

H

Core region contains unusual sugars

  • The inner core contains 1-4 KDO residues, which look like an analog of sialic acid.

  • The core also contains heptopyranoses, which can vary stereochemically

  • The rest of the core consists of various combinations of Glc and Gal

3-deoxy-D-manno-octulosonic acid (KDO)

L-glycero-D-manno-

heptulose



O antigens

O-antigen

Structure

O9

a

3

a

2

a

2

a

2

a

3

O6

a

3

b

4

b

3

a

4

b

2

O124

b

3

b

6

a

3

a

4

G

l

c

L

A

b

6

GlcLA = glucolactillic acid

O-antigens

  • O-antigens consist of 2-8 sugars, repeated ≤50 times

  • O-antigens gives rise to different serotypes and some are correlated with disease


Capsule

Gram-negative bacteria cell wall


a4

b4

a4

b4

a4

a8

a8

a8

a8

a8

b4

b3

b4

b3

b4

Capsule type

Structure

K1, polysialic acid

K5, N-acetylheparosan

Group A Streptococcus

(hyaluronan)

  • Mucoid strains contain a polysaccharide capsule

  • >80 different capsules types are known just in E. coli

  • Extraordinary diversity of structure


Mycobacteria
Mycobacteria

Crick et al. (2001) Glycobiology 11:107R




Bacterial glycoproteins
Bacterial Glycoproteins

  • Surface-layer (S-layer) glycoproteins

  • Prevalent in Bacteria and Archaea, but structures differ, e.g., N-linked glycosylation only in Archaea

  • Structural analyses have revealed unusual carbohydrate-linkage regions….

  • ….and unusual nucleoside diphosphate-linked oligosaccharides



Notice diversity of linkages

Similarity in sequence of N-linked attachment sites and mechanism of assembly of glycan (dolichol pathway)

Burda & Aebi (1999) Biochim Biophys Acta 1426:239


Yeast make asn linked glycoproteins
Yeast make AsN-linked Glycoproteins

  • Yeast make membrane N-linked glycoproteins much in the same way as higher eukaryotes

  • Serves as a model for human genetic diseases (CDG)

M Aebi (2001) Trends in Cell Biology 11:136


Yeast mannans

a 6

a 6

a 2

a 3

a 2

a 6

a 2

a 3

a 2

a 6

a 2

a 3

a 2

a 3

a 6

a 2

a 3

a 2

a 3

a 3

a 2

a 6

a 6

a 3

-1-P

a 2

a 4

a 3

a 6

a 6

-P-

a 2

a 3

a 2

a 2

a 3

a 6

a 2

a

b4

a

Ser/Thr

Ser/Thr

b4

Vertebrate

a-dystroglycan

Asn

Yeast Mannans

Willer et al (2003) Curr Opin Struct Biol 13:621


Yeast cell walls

GPI

proteins

GPI

proteins

GPI

proteins

Pir cell wall

proteins

b1,6-glucans

b1,6-glucans

b1,6-glucans

b1,3-glucans

Plasma

membrane

Yeast Cell Walls

The cell wall is made of 60% b-glucans, 40% mannoproteins, and ~1% chitin

Smits et al (2001) Microbiology 147:781


Summary
Summary

  • Bacterial cell walls are complex layered structures composed of multiple classes of glycans

  • Bacterial wall glycans provide an exoskeleton, define shape, protect against turgor pressure, create antigenic serotypes, and provide a way to prevent immune recognition

  • Glycosylated proteins are present in eubacteria and in archaea

  • Yeast produce many of the same glycans found in higher eukaryotes, but the mecahnism of assembly differs in subtle way

  • Yeast cell walls are composed of glycans (glucans and chitin)

  • Yeast and bacteria provide powerful genetic systems for understanding membrane and cell wall assembly


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